Phosphoribosylaminoimidazole carboxylase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a phosphoribosylaminoimidazole carboxylase. The invention also relates to the construction of a recombinant DNA construct or chimeric gene encoding all or a portion of the phosphoribosylaminoimidazole carboxylase, in sense or antisense orientation, wherein expression of the recombinant DNA construct or chimeric gene results in production of altered levels of the phosphoribosylaminoimidazole carboxylase in a transformed host cell.

[0001] This application claims the benefit of U.S. Provisional Application No. 60/341,955, filed Dec. 19, 2001, the entire content of which is herein incorporated by reference.

FIELD OF INVENTION

[0002] The field of invention relates to plant molecular biology, and more specifically, to nucleic acid fragments encoding phosphoribosylamino-imidazole carboxylase in plants and seeds.

BACKGROUND OF INVENTION

[0003] Purine and pyrimidine nucleotides are produced in the cell by de novo biosynthetic pathways and by salvage pathways. Salvage pathways function to recover nucleotide bases released during the degradation of nucleic acids. Purines are components of DNA and RNA. Regulation of purine synthesis and metabolism within the cell is critical to functions of all cells. Most mutations affecting nucleotide biosynthetic enzymes are lethal, although certain redundancy and salvage pathways may moderate the deleterious effects of some of these mutations. The detailed pathway of purine biosynthesis was worked out in the 1950s. The origin of carbon atom 6 of the purine ring from carbon dioxide during the synthesis of the purine ring by chicken liver was attributed to phosphoribosylaminoimidazole carboxylase (AIR carboxylase) (Lukens, L. N. and Buchanan, J. M., J. Biol. Chem. 234(7):1799-1805 (1959)). AIR carboxylase is an enzyme that functions in purine metabolism converting 5-amino-1-ribosylimidazole 5′-phosphate (AIR) and carbon dioxide to 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5′-phosphate (carboxy-AIR).

[0004] De novo synthesis of the purine, inosine monophosphate, is achieved via ten enzymatic reactions. In eukaryotic cells several of the steps are catalyzed by multifunctional proteins. However, the regulation of de novo purine biosynthesis in the nodules of tropical legumes (mothbean; Vigna aconitifolia) revealed that the AIR carboxylase (NCBI General Identification (GI) No. 1709930) and the next enzyme in the purine pathway (5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase) are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide (Chapman, K. A.; Delauney, A. J.; Kim, J. H. and Verma, D. P., Plant Mol. Biol. 24(2)389-395 (1994)). The activities and some properties of AIR carboxylase and SAICAR synthetase were also examined from wheat seedlings (Triticum aestivum) and gel filtration (no sequence information) showed that in higher plants the two were also individual proteins (Dolgikh, E. A.; Dolgikh, V. V. and Domkin, V. D., Russ. J. Plant Physiol. 43(1):10-16 (1996)).

SUMMARY OF INVENTION

[0005] The present invention includes isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having phosphoribosylaminoimidazole carboxylase activity wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17 have preferably at least 80% sequence identity. It is also preferred that the identity be at least 85%, at least 90%, or at least 95%. The present invention also includes isolated polynucleotides comprising the complement of the nucleotide sequence. More specifically, the present invention includes isolated polynucleotides encoding the polypeptide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17 or nucleotide sequences comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11 or 16.

[0006] In a first embodiment, the present invention includes an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having phosphoribosylaminoimidazole carboxylase activity, wherein the polypeptide has an amino acid sequence of at least 80%, 85%, 90%, or 95% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17, or (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. The polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17. The nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11 or 16.

[0007] In a second embodiment, the present invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, and a cell, a plant, and a seed comprising the recombinant DNA construct.

[0008] In a third embodiment, the present invention includes a vector comprising any of the isolated polynucleotides of the present invention.

[0009] In a fourth embodiment, the present invention concerns a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention. The cell transformed by this method is also included. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0010] In a fifth embodiment, the present invention includes a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a plant from the transformed plant cell, a transgenic plant produced by this method, and seed obtained from this transgenic plant.

[0011] In a sixth embodiment, the present invention includes an isolated polypeptide having phosphoribosylaminoimidazole carboxylase activity, wherein the polypeptide has an amino acid sequence of at least 80%, 85%, 90%, or 95% identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8, 10,12 or 17. The polypeptide preferably comprises one of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17.

[0012] In a seventh embodiment, the present invention includes a method for isolating a polypeptide having phosphoribosylaminoimidazole carboxylase activity comprising isolating the polypeptide from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the present invention operably linked to at least one regulatory sequence.

[0013] In an eighth embodiment, this invention includes a method for selecting a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, under conditions that allow expression of the phosphoribosylaminoimidazole carboxylase polynucleotide in an amount sufficient to complement a null mutant in order to provide a positive selection means.

[0014] In a ninth embodiment, this invention includes a method of altering the level of expression of a phosphoribosylaminoimidazole carboxylase protein in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the phosphoribosylaminoimidazole carboxylase protein in the transformed host cell.

[0015] A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a phosphoribosylaminoimidazole carboxylase, the method comprising the steps of: (a) introducing into a host cell a recombinant DNA construct comprising a nucleic acid fragment encoding a phosphoribosylaminoimidazole carboxylase polypeptide, operably linked to at least one regulatory sequence; (b) growing the host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of phosphoribosylaminoimidazole carboxylase polypeptide in the host cell; (c) optionally purifying the phosphoribosylaminoimidazole carboxylase polypeptide expressed by recombinant DNA construct in the host cell; (d) treating the phosphoribosylaminoimidazole carboxylase polypeptide with a compound to be tested; (e) comparing the activity of the phosphoribosylaminoimidazole carboxylase polypeptide that has been treated with a test compound to the activity of an untreated phosphoribosylaminoimidazole carboxylase polypeptide, and (f) selecting compounds with potential for inhibitory activity.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

[0016]FIGS. 1A and 1B depict the ten enzymatic reactions that are required for the de novo synthesis of the purine, inosine monophosphate (inosinate), from the precursor phosphoribosylpyrophosphate (PRPP). Phosphoribosylaminoimidazole (AIR) carboxylase is the sixth step and it converts 5-amino-1-ribosylimidazole 5′-phosphate (AIR) and carbon dioxide to 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5′-phosphate (carboxy-AIR).

[0017]FIGS. 2A, 2B, 2C and 2D depict the amino acid sequence alignment of the phosphoribosylaminoimidazole carboxylases encoded by the following:

[0018] (a) nucleotide sequence derived from corn clone cpd1c.pk002.l23 (SEQ ID NO:2),

[0019] (b) nucleotide sequence derived from corn clone cho1c.pk003.f1 (SEQ ID NO:4),

[0020] (c) nucleotide sequence derived from corn clone cpi1c.pk011.g12 (SEQ ID NO:6),

[0021] (d) nucleotide sequence derived from rice clone rdi2c.pk010.p22 (SEQ ID NO:8),

[0022] (e) nucleotide sequence derived from brassica clone ebp1f.pk002.g18 (SEQ ID NO:1 7), (f) nucleotide sequence from Vigna aconitifolia (NCBI General Identification (GI) No. 1709930; SEQ ID NO:15), (g) nucleotide sequence from Arabidopsis thaliana (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) and

[0023] (h) nucleotide sequence from Nicotiana tabacum (NCBI General Identification (GI) No. 13173434; SEQ ID NO:14). Dashes are used by the program to maximize alignment of the sequences.

[0024]FIG. 3 depicts the amino acid alignment of the catalytic region of Vigna aconitifolia (NCBI General Identification (GI) No. 1709930; SEQ ID NO:15) (nucleotides 387 to 557) with the amino acid sequences encoded by the following:

[0025] (a) nucleotide sequence derived from corn clone cpd1c.pk002.l 23 (SEQ ID NO:2),

[0026] (b) nucleotide sequence derived from corn clone cho1c.pk003.f1 (SEQ ID NO:4),

[0027] (c) nucleotide sequence derived from corn clone cpi1c.pk011.g12 (SEQ ID NO:6),

[0028] (d) nucleotide sequence derived from rice clone rdi2c.pk010.p22 (SEQ ID NO:8),

[0029] (e) nucleotide sequence derived from brassica clone ebp1f.pk002.g18 (SEQ ID NO:17), (f) nucleotide sequence from Arabidopsis thaliana (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) and (g) nucleotide sequence from Nicotiana tabacum (NCBI General Identification (GI) No.13173434; SEQ ID NO:14). When all the amino acids match the residue of the Consensus the residue of the Consensus will show, otherwise a “.” will show.

[0030] Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire or functional protein derived from an FIS, a contig, or an FIS and PCR fragment sequence (“CGS”). The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. TABLE 1 Phosphoribosylaminoimidazole Carboxylase SEQ ID NO: Plant Clone Designation Status (Nucleotide) (Amino Acid) Corn cpd1c.pk002.123:fis CGS 1 2 Corn cho1c.pk003.f1:fis CGS 3 4 Corn cpi1c.pk011.g12:fis FIS 5 6 Rice rdi2c.pk010.p22:fis CGS 7 8 Soybean sfl1.pk0118.d10:fis FIS 9 10 Wheat Contig of FIS 11 12 wre1n.pk0004.g6:fis Brassica ebp1f.pk002.g18:fis CGS 16 17

[0031] SEQ ID NO:13 is the amino acid sequence of Arabidopsis thaliana (NCBI General Identification (GI) No. 7436526).

[0032] SEQ ID NO:14 is the amino acid sequence of Nicotiana tabacum (NCBI General Identification (GI) No. 13173434).

[0033] SEQ ID NO:15 is the amino acid sequence of Vigna aconitifolia (NCBI General Identification (GI) No. 1709930).

[0034] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

[0036] In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11 or 16, or the complement of such sequences.

[0037] The term “isolated” refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

[0038] The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques. A “recombinant DNA construct” comprises any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence.

[0039] As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

[0040] As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein.

[0041] The “Clustal V method of alignment” corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp (1989) CABIOS. 5:151-153) and found in the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The “default parameters” are the parameters pre-set by the manufacturer of the program and for multiple alignments they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10, while for pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. After alignment of the sequences, using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table on the same program.

[0042] Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

[0043] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity, of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 or 16, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a phosphoribosylaminoimidazole carboxylase polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct or chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated recombinant DNA construct or chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.

[0044] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

[0045] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

[0046] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying related polypeptide sequences. Useful examples of percent identities are 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0047] A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0048] “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0049] “Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0050] “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign-gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0051] “Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0052] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0053] “Translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

[0054] “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0055] “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptides by the cell. “cDNA” refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA”0 refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0056] The term “operably linked” refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0057] The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

[0058] A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.

[0059] “Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0060] “Null mutant” refers here to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.

[0061] “Mature protein” or the term “mature” when used in describing a protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor protein” or the term “precursor” when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0062] A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

[0063] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0064] “Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. The term “transformation” as used herein refers to both stable transformation and transient transformation.

[0065] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

[0066] “Motifs” or “subsequences” refer to short regions of conserved sequences of nucleic acids or amino acids that comprise part of a longer sequence. For example, it is expected that such conserved subsequences would be important for function, and could be used to identify new homologues in plants. It is expected that some or all of the elements may be found in a homologue. Also, it is expected that one or two of the conserved amino acids in any given motif may differ in a true homologue.

[0067] “PCR” or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0068] The present invention includes an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising at least 100 amino acids, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17 have at least 70%, 75%, 80%, 85%, 90% or 95% identity based on the Clustal V method of alignment, or (b) the complement of the nucleotide sequence, wherein the complement and the nucleotide sequence contain the same number of nucleotides and are 100% complementary. The polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12 or 17. The nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11 or 16. The polypeptide preferably is a phosphoribosylaminoimidazole carboxylase.

[0069] This invention also includes the isolated complement of such polynucleotides, wherein the complement and the polynucleotide consist of the same number of nucleotides, and the nucleotide sequences of the complement and the polynucleotide have 100% complementarity.

[0070] Nucleic acid fragments encoding at least a portion of several phosphoribosylaminoimidazole carboxylases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0071] For example, genes encoding other phosphoribosylaminoimidazole carboxylases, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0072] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 and 16, and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.

[0073] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0074] In another embodiment, this invention includes viruses and host cells comprising either the recombinant DNA constructs or chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.

[0075] The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5′-phosphate (carboxy-AIR) in those cells. Therefore, these genes may be used in plant cells to alter the activity of de novo nucleic acid biosynthetic pathways and salvage pathways which may alter efficient growth and development of plant cells. More specifically, the genes of the instant invention may useful as a herbicide target to inhibit the formation of carboxy-AIR and other compounds further along in the pathway that may be essential for growth and development.

[0076] Overexpression of the proteins of the instant invention may be accomplished by first constructing a recombinant DNA construct or chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The recombinant DNA construct or chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant recombinant DNA construct or chimeric gene may also comprise one or more introns in order to facilitate gene expression.

[0077] Plasmid vectors comprising the instant isolated polynucleotide (or recombinant DNA construct or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the recombinant DNA construct or chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0078] For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the recombinant DNA construct or chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

[0079] It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a recombinant DNA construct or chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a recombinant DNA construct or chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense recombinant DNA constructs or chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

[0080] The polypeptides of the instant invention were shown to be constitutively expressed using microbead arrays (data not shown; Brenner et al., Nat. Biotechnol. 18: 630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. USA 97(4):1665-1670 (2000)).

[0081] Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of a specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

[0082] The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different recombinant DNA constructs or chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

[0083] In another embodiment, the present invention includes a phosphoribosylaminoimidazole carboxylase polypeptide having an amino acid sequence that is at least 70% identical, based on the Clustal V method of alignment, to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12 and 17.

[0084] The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a recombinant DNA construct or chimeric gene for production of the instant polypeptides. This recombinant DNA construct or chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded phosphoribosylaminoimidazole carboxylase. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).

[0085] Additionally, the instant polypeptides can be used as a target to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides/pesticides. This is desirable because the polypeptides described herein catalyze a step in purine biosynthesis, more specifically the synthesis of 5-amino-1-ribosyl-4-imidazolecarboxylic acid 5′-phosphate (carboxy-AIR). Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide/pesticide discovery and design.

[0086] All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0087] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0088] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0089] In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0090] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0091] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptide. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptide can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

EXAMPLES

[0092] The present invention is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0093] cDNA libraries representing mRNAs from various brassica (Brassica), corn (Zea mays), rice (Oiyza sativa), soybean (Glycine max) and wheat (Triticum aestivum) tissues were prepared. The characteristics of the libraries are described below. TABLE 2 cDNA Libraries from Brassica, Corn, Rice, Soybean and Wheat Library Tissue Clone ebp1f Brassica (Brassica) (OGU+, Cyclone cultivar ebp1f.pk002.g18:fis containing Ogura restorer) 1-2 mm immature whole bud cpd1c Corn (Zea mays L.) pooled BMS treated with cpd1c.pk002.123:fis chemicals related to protein kinases cpi1c Corn (Zea mays L.) pooled BMS treated with cpi1c.pk011.g12:fis chemicals related to biochemical compound synthesis cho1c Corn (Zea mays L., Alexho Synthetic High Oil) cho1c.pk003.f1:fis embryo 20 DAP rdi2c Rice (Otyza sativa, Nipponbare) developing rdi2c.pk010.p22:fis inflorescence at rachis branch-floral organ primordia formation sfl1 Soybean Immature Flower sfl1.pk0118.d10:fis wre1n Wheat Root From 7 Day Old Etiolated Seedling* wre1n.pk0004.g6:fis

[0094] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

[0095] Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.

[0096] Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.

[0097] Sequence data is collected (ABI Prism Collections) and assembled using Phred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).

[0098] In some of the clones the cDNA fragment corresponds to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information one of two different protocols are used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution. . In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5′-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3′-untranslated region (also referred to as UTR). In the second round of amplification a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

[0099] cDNA clones encoding phosphoribosylaminoimidazole carboxylases were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “plog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

[0100] ESTs submitted for analysis are compared to the GenBank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.

Example 3 Characterization of cDNA Clones Encoding Phosphoribosylaminoimidazole Carboxylase

[0101] The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to phosphoribosylaminoimidazole carboxylase from either Vigna aconitifolia (NCBI General Identification (GI) No. 1709930; SEQ ID NO:15), Arabidopsis thaliana (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) or Nicotiana tabacum (NCBI General Identification (GI) No.13173434; SEQ ID NO:14).

[0102] Shown in Table 3 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), the sequences of contigs assembled from two or more ESTs (“Contig”), sequences of contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding an entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”): TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to Phosphoribosylaminoimidazole Carboxylase Clone Status NCBI GI No. BLAST pLog Score ebp1f.pk002.g18:fis CGS 7436526 180.00 cpd1c.pk002.I23:fis CGS 7436526 180.00 cho1c.pk003.f1:fis CGS 13173434 180.00 cpi1c.pk011.g12:fis FIS 13173434 180.00 rdi2c.pk010.p22:fis CGS 13173434 180.00 sfl1.pk0118.d10:fis FIS 1709930 51.00 Contig of FIS 7436526 31.22 wre1n.pk0004.g6:fis

[0103] The nucleotide sequence of the entire cDNA insert in clone ebp1f.pk002.g18 is shown in SEQ ID NO:16. The amino acid sequence deduced from nucleotides 112 through 2025 of SEQ ID NO:16 is shown in SEQ ID NO:17 (stop codon encoded by nt 2026-2028). The nucleotide sequence of the entire cDNA insert in clone cpd1c.pk002.l23 is shown in SEQ ID NO:1. The amino acid sequence deduced from nucleotides 103 through 2004 of SEQ ID NO:1 is shown in SEQ ID NO:2 (stop codon encoded by nt 2005-2007). The nucleotide sequence of the entire cDNA insert in clone chol c.pk003.f1 is shown in SEQ ID NO:3. The amino acid sequence deduced from nucleotides 231 through 2138 of SEQ ID NO:3 is shown in SEQ ID NO:4 (stop codon encoded by nt 2139-2141). The nucleotide sequence of the entire cDNA insert in clone cpi1c.pk011.g12 is shown in SEQ ID NO:5. The amino acid sequence deduced from nucleotides 3 through 1571 of SEQ ID NO:5 is shown in SEQ ID NO:6 (stop codon encoded by nt 1572-1574). The nucleotide sequence of the entire cDNA insert in clone rdi2c.pk010.p22 is shown in SEQ ID NO:7. The amino acid sequence deduced from nucleotides 147 through 2039 of SEQ ID NO:7 is shown in SEQ ID NO:8 (stop codon encoded by nt 2040-2042). The nucleotide sequence of the entire cDNA insert in clone sfl1.pk0118.d10 is shown in SEQ ID NO:9. The amino acid sequence deduced from nucleotides 1 through 333 of SEQ ID NO:9 is shown in SEQ ID NO:10 (stop codon encoded by nt 334-336). The nucleotide sequence of the contig of clone wre1n.pk0004.g6 is shown in SEQ ID NO:11. The amino acid sequence deduced from nucleotides 1 through 348 of SEQ ID NO:11 is shown in SEQ ID NO:12.

[0104]FIGS. 2A, 2B, 2C and 2D present an alignment of the amino acid sequences set forth in SEQ ID Nos:2, 4, 6, 8, and 17, and the Vigna aconitifolia sequence (NCBI General Identification (GI) No.1709930; SEQ ID NO:15), Arabidopsis thaliana sequence (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) and Nicotiana tabacum sequence (NCBI General Identification (GI) No.13173434; SEQ ID NO:14). The data in Table 4 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID Nos:2, 4, 6, 8 and 17, and the Vigna aconitifolia sequence (NCBI General Identification (GI) No.1709930; SEQ ID NO:15), Arabidopsis thaliana sequence (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) or Nicotiana tabacum sequence (NCBI General Identification (GI) No. 13173434; SEQ ID NO:14). TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Phosphoribosylaminoimidazole Carboxylase Percent Identity to SEQ ID NOBI GI No. 1709930 NOBI GI No. 7436526 NOBI GI No. 13173434 NO. (SEQ ID NO: 15) (SEQ ID NO: 13) (SEQ ID NQ: 14) 17 71.8 89.4 79.4 2 60.8 64.7 69.4 4 60.2 64.1 68.8 6 59.6 63.5 68.2 8 59.6 64.1 66.5

[0105]FIG. 3 depicts the amino acid alignment of the catalytic region of Vigna aconitifolia (NCBI General Identification (GI) No. 1709930; SEQ ID NO:15) (nucleotides 387 to 557) with the amino acid sequences encoded by the following:

[0106] (a) nucleotide sequence derived from corn clone cpd1c.pk002.l23 (SEQ ID NO:2),

[0107] (b) nucleotide sequence derived from corn clone cho1c.pk003.f1 (SEQ ID NO:4),

[0108] (c) nucleotide sequence derived from corn clone cpi1c.pk011.g12 (SEQ ID NO:6),

[0109] (d) nucleotide sequence derived from rice clone rdi2c.pk010.p22 (SEQ ID NO:8),

[0110] (e) nucleotide sequence derived from brassica clone ebp1f.pk002.g18 (SEQ ID NO:17), (f) nucleotide sequence from Arabidopsis thaliana (NCBI General Identification (GI) No. 7436526; SEQ ID NO:13) and (g) nucleotide sequence from Nicotiana tabacum (NCBI General Identification (GI) No. 13173434; SEQ ID NO: 14).

[0111] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a phosphoribosylaminoimidazole carboxylase. These sequences represent the first monocot species sequences encoding phosphoribosylaminoimidazole carboxylase known to Applicant.

Example 4 Expression of Recombinant DNA Constructs or Chimeric Genes in Monocot Cells

[0112] A recombinant DNA construct or chimeric gene comprising a cDNA encoding the instant polypeptide in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SaII-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SaII fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a recombinant DNA construct or chimeric gene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3′ region.

[0113] The recombinant DNA construct or chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0114] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0115] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0116] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0117] Seven days after bombardment the tissue can be transferred to N6 medium that contains bialophos (5 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing bialophos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialophos-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0118] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Recombinant DNA Constructs or Chimeric Genes in Dicot Cells

[0119] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

[0120] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

[0121] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0122] Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0123] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0124] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptide and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0125] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0126] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0127] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 6 Expression of Recombinant DNA Constructs or Chimeric Genes in Microbial Cells

[0128] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0129] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0130] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25° C. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 7 Evaluating Compounds for Their Ability to Inhibit the Activity of Phosphoribosylaminoimidazole Carboxylase

[0131] The polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines. The instant polypeptides may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags. Common fusion protein partners include glutathione S-transferase (“GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide (“(His)₆”). The fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme. Examples of such proteases include thrombin, enterokinase and factor Xa. However, any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.

[0132] Purification of the instant polypeptides, if desired, may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor. When the instant polypeptides are expressed as fusion proteins, the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme. For example, the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin. In addition, a (His)₆ peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification. Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B. In an alternate embodiment, a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include β-mercaptoethanol or other reduced thiol. The eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired. Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBond™ affinity resin or other resin.

[0133] Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the instant polypeptides disclosed herein. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for phosphoribosylaminoimidazole carboxylase are presented by Lukens, L. N. and Buchanan, J. M., J. Biol. Chem. 234(7):1799-1805 (1959)).

1 17 1 2199 DNA Zea mays 1 gcacgaggca cgagcggcac gagctagaca cccatcgtct cccgttcccg gctaaaccct 60 gcttgagccc tagacaccgc ccgcctccgg gaggtagaga gcatgcacgc caggttcctc 120 agcgcgccgt cccctgcctc cgccgccccc tccccgtacg ttcgcttggc cttcacgggc 180 gcccgcccgc gccgcgcgtg ttggaagccg cgaggccccg cgtccgcgtc cgcgccgccg 240 cctcggcctc tccactcgct gtgcgcccgg gcctccatgc agcccgcctc tcccgcgcac 300 gacgggcatg gtggtccgcc ggtgcacggc gtctccaaca ccgtcatcgg ggtcctgggg 360 ggcggtcagc tcgggaagat gctctgccat gcagcgagtc aaatggggat tagaattgtc 420 atcctcgacc ctcttcccgg ctgccccgcg agctcggttt gcgatgagca cgtaatcggg 480 agcttcaacg atagggacac ggtccgggag ttcgccaaga ggtgtggggt cctaacagtg 540 gagattgagc atgttgatgc cgccacactg gagaagctgg aaaaacaggg cgttgactgc 600 gagcctaaag cctccacaat cacgattatt caggacaagt acaggcagaa aaagcatttc 660 tcaagatgcg agatcccatt gcctgacttt atggaagtag atactttacg cagtatagag 720 gaggctgggg aaaagtttgg ctatccccta atggtcaaaa gcaagagatt agcatatgat 780 ggtcgaggaa atgctgtagc caagaacaga gaggagctac cttctgttgt tgcttcactg 840 ggtgggtttg agcggggctt gtatgttgag agatggactc ctttcgtaaa ggagctttct 900 gtaattgtgg caaggagcag agacaactct actgtctgct atcctgttgt ggaaacagtt 960 cacaaggaaa atatatgcca tgttgttgaa gctcctgctg atttatctaa caaaataaag 1020 aagttagcta ctagcgtggc tgaaaaagct atcaaatcat tagaaggagc tggtgtcttt 1080 gctgtagagt tgtttttaac agaagatgat cagattttat tgaatgaggt agcccctagg 1140 cctcacaata gtgggcatca cacaatagag tcatgctaca cctcacaata tgagcagcat 1200 atacgcgcta ttcttggcct tcctcttggt gatccctcaa tgaaagcacc tgcagcaata 1260 atgtacaaca tcctgggcga ggatgagggt gaagcagggt tctttctggc tcatcagctt 1320 atcagtaggg cactaaccat tccaggcaca tcggtccatt ggtacgcaaa gccagagatg 1380 cggaagcaaa ggaagatggg tcatattaca attgtggggc cttctaagat aagtgtaaaa 1440 tcacgcttgg acaacttgct gcaaagaaac tcgtctgatc ccaaggaagt tagccctcgt 1500 gttgctatta taatggggtc ccaatctgat cttcctgtga tgaaagatgc tgagagggtt 1560 ttgaaagagt tcgacatacc ttgtgaggta actattgttt ctgcacatcg tacaccagag 1620 cggatgtatg attatgcgaa gtctgctaaa gacaggggtt tcgaggtcat aattgcaggt 1680 gcaggcggag cagctcattt accagggatg gtggcttcat tgactcctct tcctgtaatc 1740 ggagttccca ttaagacttc aacactatca ggatttgatt ccctcctatc tattgtgcaa 1800 atgccaaaag gtattcctgt tgcgactgtt gctatcggga atgcagaaaa tgcaggtttg 1860 ctggcagcta ggattctggc tgcaagagat cctgagctcc aggacagggt aactaagtac 1920 caggatgatc tgagggacat ggttttggag acggcagaaa ggctggagga ccaaggcccg 1980 gaggaatttc tgaagggaat ggattgaccc cttttgaagc cctggctctg gggcctggga 2040 gaggaatacg agtgtatggg tcggcgataa ctcactgtcc actcgattat ggtttaagat 2100 ggggagtcat caagaagata ctttaatagg ctcggctgca ttcgattttc cttacaattc 2160 ttttttgata aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2199 2 634 PRT Zea mays 2 Met His Ala Arg Phe Leu Ser Ala Pro Ser Pro Ala Ser Ala Ala Pro 1 5 10 15 Ser Pro Tyr Val Arg Leu Ala Phe Thr Gly Ala Arg Pro Arg Arg Ala 20 25 30 Cys Trp Lys Pro Arg Gly Pro Ala Ser Ala Ser Ala Pro Pro Pro Arg 35 40 45 Pro Leu His Ser Leu Cys Ala Arg Ala Ser Met Gln Pro Ala Ser Pro 50 55 60 Ala His Asp Gly His Gly Gly Pro Pro Val His Gly Val Ser Asn Thr 65 70 75 80 Val Ile Gly Val Leu Gly Gly Gly Gln Leu Gly Lys Met Leu Cys His 85 90 95 Ala Ala Ser Gln Met Gly Ile Arg Ile Val Ile Leu Asp Pro Leu Pro 100 105 110 Gly Cys Pro Ala Ser Ser Val Cys Asp Glu His Val Ile Gly Ser Phe 115 120 125 Asn Asp Arg Asp Thr Val Arg Glu Phe Ala Lys Arg Cys Gly Val Leu 130 135 140 Thr Val Glu Ile Glu His Val Asp Ala Ala Thr Leu Glu Lys Leu Glu 145 150 155 160 Lys Gln Gly Val Asp Cys Glu Pro Lys Ala Ser Thr Ile Thr Ile Ile 165 170 175 Gln Asp Lys Tyr Arg Gln Lys Lys His Phe Ser Arg Cys Glu Ile Pro 180 185 190 Leu Pro Asp Phe Met Glu Val Asp Thr Leu Arg Ser Ile Glu Glu Ala 195 200 205 Gly Glu Lys Phe Gly Tyr Pro Leu Met Val Lys Ser Lys Arg Leu Ala 210 215 220 Tyr Asp Gly Arg Gly Asn Ala Val Ala Lys Asn Arg Glu Glu Leu Pro 225 230 235 240 Ser Val Val Ala Ser Leu Gly Gly Phe Glu Arg Gly Leu Tyr Val Glu 245 250 255 Arg Trp Thr Pro Phe Val Lys Glu Leu Ser Val Ile Val Ala Arg Ser 260 265 270 Arg Asp Asn Ser Thr Val Cys Tyr Pro Val Val Glu Thr Val His Lys 275 280 285 Glu Asn Ile Cys His Val Val Glu Ala Pro Ala Asp Leu Ser Asn Lys 290 295 300 Ile Lys Lys Leu Ala Thr Ser Val Ala Glu Lys Ala Ile Lys Ser Leu 305 310 315 320 Glu Gly Ala Gly Val Phe Ala Val Glu Leu Phe Leu Thr Glu Asp Asp 325 330 335 Gln Ile Leu Leu Asn Glu Val Ala Pro Arg Pro His Asn Ser Gly His 340 345 350 His Thr Ile Glu Ser Cys Tyr Thr Ser Gln Tyr Glu Gln His Ile Arg 355 360 365 Ala Ile Leu Gly Leu Pro Leu Gly Asp Pro Ser Met Lys Ala Pro Ala 370 375 380 Ala Ile Met Tyr Asn Ile Leu Gly Glu Asp Glu Gly Glu Ala Gly Phe 385 390 395 400 Phe Leu Ala His Gln Leu Ile Ser Arg Ala Leu Thr Ile Pro Gly Thr 405 410 415 Ser Val His Trp Tyr Ala Lys Pro Glu Met Arg Lys Gln Arg Lys Met 420 425 430 Gly His Ile Thr Ile Val Gly Pro Ser Lys Ile Ser Val Lys Ser Arg 435 440 445 Leu Asp Asn Leu Leu Gln Arg Asn Ser Ser Asp Pro Lys Glu Val Ser 450 455 460 Pro Arg Val Ala Ile Ile Met Gly Ser Gln Ser Asp Leu Pro Val Met 465 470 475 480 Lys Asp Ala Glu Arg Val Leu Lys Glu Phe Asp Ile Pro Cys Glu Val 485 490 495 Thr Ile Val Ser Ala His Arg Thr Pro Glu Arg Met Tyr Asp Tyr Ala 500 505 510 Lys Ser Ala Lys Asp Arg Gly Phe Glu Val Ile Ile Ala Gly Ala Gly 515 520 525 Gly Ala Ala His Leu Pro Gly Met Val Ala Ser Leu Thr Pro Leu Pro 530 535 540 Val Ile Gly Val Pro Ile Lys Thr Ser Thr Leu Ser Gly Phe Asp Ser 545 550 555 560 Leu Leu Ser Ile Val Gln Met Pro Lys Gly Ile Pro Val Ala Thr Val 565 570 575 Ala Ile Gly Asn Ala Glu Asn Ala Gly Leu Leu Ala Ala Arg Ile Leu 580 585 590 Ala Ala Arg Asp Pro Glu Leu Gln Asp Arg Val Thr Lys Tyr Gln Asp 595 600 605 Asp Leu Arg Asp Met Val Leu Glu Thr Ala Glu Arg Leu Glu Asp Gln 610 615 620 Gly Pro Glu Glu Phe Leu Lys Gly Met Asp 625 630 3 1838 DNA Zea mays 3 gcacgagctg ccccgcgagc tcggtttgcg atgagcacgt cattgggagc ttcaatgatg 60 aggacacggt ccgggagttc gccaagaggt gtggggtcct aacagtggag attgagcatg 120 ttgatgtcac cacactggag aaactggaaa aacagggcgt tgactgcgag cctaacgcct 180 ccacaatcat gattattcag gacaagtaca ggcagaaaaa gcatttctca agatgcgaga 240 tcccattgcc tgacttcatg gaagtagata ctttacgcag tatagaggag gctggagaaa 300 agtttggcta tcccctaatg gtcaaaagca agagattagc atatgatggt cgaggaaatg 360 ctgtagccaa gaacaaagag gagctacctt ctgttgttgc ttcactaggt gggtttgagc 420 ggggcttgta tgttgagaga tggactcctt tcgtaaagga gctttctgta atcgtggcaa 480 ggagcagaga caactctact gtctgctatc ctgttgtgga aacagttcac aaggaaaata 540 tatgccatgt tgttgaagct cctgctgatg tatctaacaa aataaagaag ttagctacta 600 gcgtggctga aaaagctatt aaatcattag aaggagctgg tgtctttgct gtagagttgt 660 ttttaacaga agatgatcag gttttattga atgaggtagc ccctaggcct cacaatagtg 720 gacatcacac aatagagtca tgctacacct cacaatatga gcagcatata cgcgctattc 780 ttggccttcc tcttggtgat ccctcaatga aagcacctgc agcaataatg tacaacatcc 840 tgggcgagga tgagggtgaa gcagggttct ttctggctca tcagcttatc agtagggcac 900 taaccattcc aggcacatcg gtccattggt acgcaaagcc agagatgcgg aagcaaagga 960 agatgggtca tattacaatt gtggggcctt ctaagataag tgtaaaatca cgcttggaca 1020 acttgctgca aagaaactcg tctgatccca aggaagttag ccctcgtgtt gctattataa 1080 tggggtccca atctgatctt cctgtgatga aagatgctga gagggttttg aaagagttcg 1140 acataccttg tgagctaact attgtttctg cacatcgtac accagagcgg atgtatgatt 1200 atgcgaagtc tgctaaagac aggggtttcg aggtcataat tgcaggtgca ggcggagcag 1260 ctcatttacc agggatggtg gcttcattga ctcctcttcc tgtaatcgga gttcccatta 1320 agacttcaac actatcagga tttgattccc tcctatctat tgtgcaaatg ccaaaaggta 1380 ttcctgttgc gactgttgct atcgggattg cagaaaatgc aggtttgctg gcagctagga 1440 ttctggctgc aagagatcct gagctccagg acagggtaac taagtaccag gatgatctga 1500 gggacatggt tttggagaca gcagaaaggc tggaggacca aggcccggag gaatttctga 1560 agggaatgga ttgacccctt ttgaagccct ggctctgggg cctgggagga atacgaatat 1620 atgggtcggc gataactcag tccactcgat tatggtttaa gatcgggagt catcaaaaag 1680 atactttaag gactagttcc ggagctacaa aacctgaggg gattgggggc tagaatctcc 1740 gtgactctta gagctgtttt gatgaccaag aatcacgaag ggatccatag gcttggctgc 1800 cttattcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1838 4 523 PRT Zea mays 4 Thr Ser Cys Pro Ala Ser Ser Val Cys Asp Glu His Val Ile Gly Ser 1 5 10 15 Phe Asn Asp Glu Asp Thr Val Arg Glu Phe Ala Lys Arg Cys Gly Val 20 25 30 Leu Thr Val Glu Ile Glu His Val Asp Val Thr Thr Leu Glu Lys Leu 35 40 45 Glu Lys Gln Gly Val Asp Cys Glu Pro Asn Ala Ser Thr Ile Met Ile 50 55 60 Ile Gln Asp Lys Tyr Arg Gln Lys Lys His Phe Ser Arg Cys Glu Ile 65 70 75 80 Pro Leu Pro Asp Phe Met Glu Val Asp Thr Leu Arg Ser Ile Glu Glu 85 90 95 Ala Gly Glu Lys Phe Gly Tyr Pro Leu Met Val Lys Ser Lys Arg Leu 100 105 110 Ala Tyr Asp Gly Arg Gly Asn Ala Val Ala Lys Asn Lys Glu Glu Leu 115 120 125 Pro Ser Val Val Ala Ser Leu Gly Gly Phe Glu Arg Gly Leu Tyr Val 130 135 140 Glu Arg Trp Thr Pro Phe Val Lys Glu Leu Ser Val Ile Val Ala Arg 145 150 155 160 Ser Arg Asp Asn Ser Thr Val Cys Tyr Pro Val Val Glu Thr Val His 165 170 175 Lys Glu Asn Ile Cys His Val Val Glu Ala Pro Ala Asp Val Ser Asn 180 185 190 Lys Ile Lys Lys Leu Ala Thr Ser Val Ala Glu Lys Ala Ile Lys Ser 195 200 205 Leu Glu Gly Ala Gly Val Phe Ala Val Glu Leu Phe Leu Thr Glu Asp 210 215 220 Asp Gln Val Leu Leu Asn Glu Val Ala Pro Arg Pro His Asn Ser Gly 225 230 235 240 His His Thr Ile Glu Ser Cys Tyr Thr Ser Gln Tyr Glu Gln His Ile 245 250 255 Arg Ala Ile Leu Gly Leu Pro Leu Gly Asp Pro Ser Met Lys Ala Pro 260 265 270 Ala Ala Ile Met Tyr Asn Ile Leu Gly Glu Asp Glu Gly Glu Ala Gly 275 280 285 Phe Phe Leu Ala His Gln Leu Ile Ser Arg Ala Leu Thr Ile Pro Gly 290 295 300 Thr Ser Val His Trp Tyr Ala Lys Pro Glu Met Arg Lys Gln Arg Lys 305 310 315 320 Met Gly His Ile Thr Ile Val Gly Pro Ser Lys Ile Ser Val Lys Ser 325 330 335 Arg Leu Asp Asn Leu Leu Gln Arg Asn Ser Ser Asp Pro Lys Glu Val 340 345 350 Ser Pro Arg Val Ala Ile Ile Met Gly Ser Gln Ser Asp Leu Pro Val 355 360 365 Met Lys Asp Ala Glu Arg Val Leu Lys Glu Phe Asp Ile Pro Cys Glu 370 375 380 Leu Thr Ile Val Ser Ala His Arg Thr Pro Glu Arg Met Tyr Asp Tyr 385 390 395 400 Ala Lys Ser Ala Lys Asp Arg Gly Phe Glu Val Ile Ile Ala Gly Ala 405 410 415 Gly Gly Ala Ala His Leu Pro Gly Met Val Ala Ser Leu Thr Pro Leu 420 425 430 Pro Val Ile Gly Val Pro Ile Lys Thr Ser Thr Leu Ser Gly Phe Asp 435 440 445 Ser Leu Leu Ser Ile Val Gln Met Pro Lys Gly Ile Pro Val Ala Thr 450 455 460 Val Ala Ile Gly Ile Ala Glu Asn Ala Gly Leu Leu Ala Ala Arg Ile 465 470 475 480 Leu Ala Ala Arg Asp Pro Glu Leu Gln Asp Arg Val Thr Lys Tyr Gln 485 490 495 Asp Asp Leu Arg Asp Met Val Leu Glu Thr Ala Glu Arg Leu Glu Asp 500 505 510 Gln Gly Pro Glu Glu Phe Leu Lys Gly Met Asp 515 520 5 2434 DNA Zea mays 5 gcaccagaca aatcgctttc cgcgccgccg ccgcgccgcc gacctagaca cccatcgtct 60 cccgttcccg gctaaaccct gcttgagccc tagacaccgc ccgcctccgg gaggtagaga 120 gcatgcacgc caggttcctc agcgcgccgt cccctgcctc ccggaggctg atgagccggc 180 taaaccctgc ttgagcccta gacaccgccc gcctccggga ggtagagagc atgcacgcca 240 ggttcctcag cgcgccgtcc cctgcctccg ccgccccctc cccgtacgtt cgcttggcct 300 tcacgggcgc ccgcccgcgc cgcgcgtgtt ggaagccggg gccgcgaggc cccgcgtccg 360 cgtccgcgcc gccgcctcgg cctctccact cgctgtgcgc ccgggcctcc atgcaggccg 420 cctctcccgc gcacgacggg catggtggtc cgccggtgca cggcgtctcc aacaccgtca 480 tcggggtcct ggggggcggt cagctcggga agatgctctg ccatgcagcg agtcagatgg 540 ggattagaat tgtcatcctc gaccctcttc ccggatgccc cgcgagctcg gtttgcgatg 600 agcacgtcat cgggagcttc aacgatgggg acacggtccg ggagttcgcc aagaggtgtg 660 gggtcctaac agtggaaatt gagcatgttg atgccgccac actggagaag ctggaaaaac 720 agggcgttga ctgcgagcct aaagcctcca caatcacgat tattcaggac aagtacaggc 780 agaaaaagca tttctcaaga tgcgagatcc cattgcctga cttcatggaa gtagatactt 840 tacgcattat agaggaggct ggagaaaagt ttggctatcc cctaatggtc aaaagcaaga 900 gattagcata tgatggtcga ggaaatgctg tagccaagaa caaagaggag ctaccttctg 960 ttgttgcttc actgggtggg tttgagcggg gcttgtatgt tgagagatgg actcctttcg 1020 taaaggagct ttctgtaatt gtggcaagga gcagagacaa ctctactgtc tgctatcctg 1080 ttgtggaaac agttcacaag gaaaatatat gccatgttgt tgaagctcca gctgatgtat 1140 ctaacaaaat aaataagtta gctactagcg tggctgaaaa agctatcaaa tcattagaag 1200 gagctggtgt ctttgctgta gagttgtttt taacagaaga tgatcagatt ttattgaatg 1260 aggtagcccc taggcctcac aatagtgggc atcacacaat agagtcatgc tacacctcac 1320 aatatgagca gcatatacgc gctattcttg gccttcctct tggtgatccc tcaatgaaag 1380 cacctgcagc aataatgtac aacatcctgg gcgaggatga gggtgaagca gggttctttc 1440 tggctcatca gcttatcagt agggcactaa ccattccagg cacatcggtc cattggtacg 1500 caaagccaga gatgcggaag caaaggaaga tgggtcatat tacaattgtg gggccttcta 1560 agataagtgt aaaatcacgc ttggacaact tgctgcaaag aaactcgtct gatcccaagg 1620 aagttagccc tcgtgttgct attataatgg ggtcccaatc tgatcttcct gtgatgaaag 1680 atgctgagag ggttttgaaa gagttcgaca taccttgtga gttaactatt gtttctgcac 1740 atcgtacacc agagcggatg tatgattatg cgaagtctgc taaagacagg ggtttcgagg 1800 tcataattgc aggtgcaggc ggagcagctc atttaccagg gatggtggct tcattgactc 1860 ctcttcctgt aatcggagtt cccattaaga cttcaacact atcaggattt gattccctcc 1920 tatctattgt gcaaatgcca aaaggtattc ctgttgcgac tgttgctatc gggaatgcag 1980 aaaatgcagg tttgctggca gctaggattc tggctgcaag agatcctgag ctccaggaca 2040 gggtaactaa gtaccaggat gatctgaggg acatggtttt ggagacggca gaaaggctgg 2100 aggaccaagg cccggaggaa tttctgaagg gaatggattg accccttttg aagccctggc 2160 tctggggcct gggaggaata cgaatgtatg ggtcggcgat aactcactgt ccactcgatt 2220 atggtttaag atcgggagtc atcaagaaga tactttaata ggctcggctg cattcgattt 2280 tccttacaat tcttttttga taattgtgct atcttaaccc atctcaacat gccctttttc 2340 ccttctaata tagggtatga attgggttta gagggggaaa aaagaatggg agtaaacgtt 2400 gtacgtacga aaaataaaac ggttcgagta acgt 2434 6 636 PRT Zea mays 6 Met His Ala Arg Phe Leu Ser Ala Pro Ser Pro Ala Ser Ala Ala Pro 1 5 10 15 Ser Pro Tyr Val Arg Leu Ala Phe Thr Gly Ala Arg Pro Arg Arg Ala 20 25 30 Cys Trp Lys Pro Gly Pro Arg Gly Pro Ala Ser Ala Ser Ala Pro Pro 35 40 45 Pro Arg Pro Leu His Ser Leu Cys Ala Arg Ala Ser Met Gln Ala Ala 50 55 60 Ser Pro Ala His Asp Gly His Gly Gly Pro Pro Val His Gly Val Ser 65 70 75 80 Asn Thr Val Ile Gly Val Leu Gly Gly Gly Gln Leu Gly Lys Met Leu 85 90 95 Cys His Ala Ala Ser Gln Met Gly Ile Arg Ile Val Ile Leu Asp Pro 100 105 110 Leu Pro Gly Cys Pro Ala Ser Ser Val Cys Asp Glu His Val Ile Gly 115 120 125 Ser Phe Asn Asp Gly Asp Thr Val Arg Glu Phe Ala Lys Arg Cys Gly 130 135 140 Val Leu Thr Val Glu Ile Glu His Val Asp Ala Ala Thr Leu Glu Lys 145 150 155 160 Leu Glu Lys Gln Gly Val Asp Cys Glu Pro Lys Ala Ser Thr Ile Thr 165 170 175 Ile Ile Gln Asp Lys Tyr Arg Gln Lys Lys His Phe Ser Arg Cys Glu 180 185 190 Ile Pro Leu Pro Asp Phe Met Glu Val Asp Thr Leu Arg Ile Ile Glu 195 200 205 Glu Ala Gly Glu Lys Phe Gly Tyr Pro Leu Met Val Lys Ser Lys Arg 210 215 220 Leu Ala Tyr Asp Gly Arg Gly Asn Ala Val Ala Lys Asn Lys Glu Glu 225 230 235 240 Leu Pro Ser Val Val Ala Ser Leu Gly Gly Phe Glu Arg Gly Leu Tyr 245 250 255 Val Glu Arg Trp Thr Pro Phe Val Lys Glu Leu Ser Val Ile Val Ala 260 265 270 Arg Ser Arg Asp Asn Ser Thr Val Cys Tyr Pro Val Val Glu Thr Val 275 280 285 His Lys Glu Asn Ile Cys His Val Val Glu Ala Pro Ala Asp Val Ser 290 295 300 Asn Lys Ile Asn Lys Leu Ala Thr Ser Val Ala Glu Lys Ala Ile Lys 305 310 315 320 Ser Leu Glu Gly Ala Gly Val Phe Ala Val Glu Leu Phe Leu Thr Glu 325 330 335 Asp Asp Gln Ile Leu Leu Asn Glu Val Ala Pro Arg Pro His Asn Ser 340 345 350 Gly His His Thr Ile Glu Ser Cys Tyr Thr Ser Gln Tyr Glu Gln His 355 360 365 Ile Arg Ala Ile Leu Gly Leu Pro Leu Gly Asp Pro Ser Met Lys Ala 370 375 380 Pro Ala Ala Ile Met Tyr Asn Ile Leu Gly Glu Asp Glu Gly Glu Ala 385 390 395 400 Gly Phe Phe Leu Ala His Gln Leu Ile Ser Arg Ala Leu Thr Ile Pro 405 410 415 Gly Thr Ser Val His Trp Tyr Ala Lys Pro Glu Met Arg Lys Gln Arg 420 425 430 Lys Met Gly His Ile Thr Ile Val Gly Pro Ser Lys Ile Ser Val Lys 435 440 445 Ser Arg Leu Asp Asn Leu Leu Gln Arg Asn Ser Ser Asp Pro Lys Glu 450 455 460 Val Ser Pro Arg Val Ala Ile Ile Met Gly Ser Gln Ser Asp Leu Pro 465 470 475 480 Val Met Lys Asp Ala Glu Arg Val Leu Lys Glu Phe Asp Ile Pro Cys 485 490 495 Glu Leu Thr Ile Val Ser Ala His Arg Thr Pro Glu Arg Met Tyr Asp 500 505 510 Tyr Ala Lys Ser Ala Lys Asp Arg Gly Phe Glu Val Ile Ile Ala Gly 515 520 525 Ala Gly Gly Ala Ala His Leu Pro Gly Met Val Ala Ser Leu Thr Pro 530 535 540 Leu Pro Val Ile Gly Val Pro Ile Lys Thr Ser Thr Leu Ser Gly Phe 545 550 555 560 Asp Ser Leu Leu Ser Ile Val Gln Met Pro Lys Gly Ile Pro Val Ala 565 570 575 Thr Val Ala Ile Gly Asn Ala Glu Asn Ala Gly Leu Leu Ala Ala Arg 580 585 590 Ile Leu Ala Ala Arg Asp Pro Glu Leu Gln Asp Arg Val Thr Lys Tyr 595 600 605 Gln Asp Asp Leu Arg Asp Met Val Leu Glu Thr Ala Glu Arg Leu Glu 610 615 620 Asp Gln Gly Pro Glu Glu Phe Leu Lys Gly Met Asp 625 630 635 7 2163 DNA Oryza sativa 7 gcacgaggga cgcgtcgctt tcctccgccg ccgccgcctc ctcctccgct ccgcgcggcc 60 gtctcccacc tctcgcgcct cacctcctcc tcctcctaaa ccctcgctaa accctagcct 120 cccacccccc caccccaccg gggagcatgc actccaggct cctcagcgcc ccctcccacg 180 cctccgccgc ctcctcctcc cccctcccct tcgccgccgc ccacccttgc cgcgcctcct 240 ggacgccgcg gcccacctcc ccgctgccgc ctctatcgct gctgcgcgcc accgcctcca 300 tgcatccttc tcctcctccc gaggggcata gcgaccagcc tgtgcatggc gtcaccaaca 360 cggtcgtcgg cgtgctgggg ggaggccagc tggggaagat gctgtgccag gcggccagcc 420 agatgggggt caggatggcc atacttgatc ccctcgagga ctgcccggcg agctcggttt 480 gccacgagca tgtcgtcggg agcttcaatg atggcgccac ggttagcgag ttcgcaaaga 540 ggtgcggggt tttgacggtg gaaattgagc atgtcgacgc tgtaacactc gagaagcttg 600 agaaacaggg catcgattgt gagcccaaag cctccaccat catgattatt caagacaagt 660 acaggcagaa gactcatttc tcaaaatttg gaattccgtt acctgacttt gtggaagtag 720 atactttaag tagcatagag aaagctgggg aaatgtttgg ttatcctcta atggtcaaaa 780 gcaagagatt agcatatgat ggccgtggaa atgctgttgc tcacgacaaa aaagagctat 840 cttctgttgt tgcttctctt ggtgggtttg agcatggctt gtatgttgag aggtggacat 900 cttttgtaaa ggagctttct gtcattgtgg caaggagcag agacggttct acggtgtgct 960 atcctgttgt tgaaaccatc cacaaggata acatctgcca tgttgttgag gctcctgccg 1020 aggtgcctga taaaataaag aagttggcta ctaatgtagc tgaaaaggct atcaaatcat 1080 tggaaggtgc tggtgttttc gctgtagaat tatttttaac acaagataat caggttttat 1140 tgaatgaagt agctccaagg cctcacaaca gtgggcatca cacaattgag tcatgttata 1200 cctcgcaata tgagcaacat ttacgtgcta ttcttggcct tcctcttggt gatccttcaa 1260 tgaaagcgcc tgcatcaata atgtacaaca tcctgggtga ggatgagggt gaggcaggat 1320 ttactcaagc tcatcagttg attgagagag ctttggacat ttcaggtgca tctgtccatt 1380 ggtatgcaaa accagaaata cggaagcaga gaaagatggg ccatattaca attgtggggc 1440 cttcaaagta cagtgtaaaa gcacgcttag ataagttgct gcaaagagac gcatatgacc 1500 ccaagaaagt tgcagttaaa cctcgtgctg caataataat gggttctgat tctgatcttc 1560 ctgtcatgaa agatgctgca gtagtattga agaaattcaa catacctttt gagcttacaa 1620 ttgtttcggc tcatcgtaca ccagagagga tgtaccatta tgcattatct actaaagaaa 1680 gaggcttaga ggtcataatt gcaggtgcag gtggagcggc tcacttacca gggatggtgg 1740 cttcattgac ttctgtccca gtaataggag tacccatcat gacttcatct ttacatggaa 1800 ctgattccct cctatctatt gtccagatgc cgaaaggtat tcctgttgct actgttgcaa 1860 ttggaaatgc ggaaaatgca ggtttattgg cagttaggat gctggcctca agagatcctg 1920 agttggggga caaggcaact gaataccagc atgatctgag ggatatggtg ttggagaaag 1980 caaaaaggct cgaggaacta ggttgggagg aatataccga gctatacttg aagaagcatt 2040 gatctctgct gcccgtttaa ttgacattct ttttgatcca gtgttgacat aagcagatat 2100 ggtccatggt tgtggcaaat tcttaaaatc cttttcattc ttttcaaaaa aaaaaaaaaa 2160 aaa 2163 8 631 PRT Oryza sativa 8 Met His Ser Arg Leu Leu Ser Ala Pro Ser His Ala Ser Ala Ala Ser 1 5 10 15 Ser Ser Pro Leu Pro Phe Ala Ala Ala His Pro Cys Arg Ala Ser Trp 20 25 30 Thr Pro Arg Pro Thr Ser Pro Leu Pro Pro Leu Ser Leu Leu Arg Ala 35 40 45 Thr Ala Ser Met His Pro Ser Pro Pro Pro Glu Gly His Ser Asp Gln 50 55 60 Pro Val His Gly Val Thr Asn Thr Val Val Gly Val Leu Gly Gly Gly 65 70 75 80 Gln Leu Gly Lys Met Leu Cys Gln Ala Ala Ser Gln Met Gly Val Arg 85 90 95 Met Ala Ile Leu Asp Pro Leu Glu Asp Cys Pro Ala Ser Ser Val Cys 100 105 110 His Glu His Val Val Gly Ser Phe Asn Asp Gly Ala Thr Val Ser Glu 115 120 125 Phe Ala Lys Arg Cys Gly Val Leu Thr Val Glu Ile Glu His Val Asp 130 135 140 Ala Val Thr Leu Glu Lys Leu Glu Lys Gln Gly Ile Asp Cys Glu Pro 145 150 155 160 Lys Ala Ser Thr Ile Met Ile Ile Gln Asp Lys Tyr Arg Gln Lys Thr 165 170 175 His Phe Ser Lys Phe Gly Ile Pro Leu Pro Asp Phe Val Glu Val Asp 180 185 190 Thr Leu Ser Ser Ile Glu Lys Ala Gly Glu Met Phe Gly Tyr Pro Leu 195 200 205 Met Val Lys Ser Lys Arg Leu Ala Tyr Asp Gly Arg Gly Asn Ala Val 210 215 220 Ala His Asp Lys Lys Glu Leu Ser Ser Val Val Ala Ser Leu Gly Gly 225 230 235 240 Phe Glu His Gly Leu Tyr Val Glu Arg Trp Thr Ser Phe Val Lys Glu 245 250 255 Leu Ser Val Ile Val Ala Arg Ser Arg Asp Gly Ser Thr Val Cys Tyr 260 265 270 Pro Val Val Glu Thr Ile His Lys Asp Asn Ile Cys His Val Val Glu 275 280 285 Ala Pro Ala Glu Val Pro Asp Lys Ile Lys Lys Leu Ala Thr Asn Val 290 295 300 Ala Glu Lys Ala Ile Lys Ser Leu Glu Gly Ala Gly Val Phe Ala Val 305 310 315 320 Glu Leu Phe Leu Thr Gln Asp Asn Gln Val Leu Leu Asn Glu Val Ala 325 330 335 Pro Arg Pro His Asn Ser Gly His His Thr Ile Glu Ser Cys Tyr Thr 340 345 350 Ser Gln Tyr Glu Gln His Leu Arg Ala Ile Leu Gly Leu Pro Leu Gly 355 360 365 Asp Pro Ser Met Lys Ala Pro Ala Ser Ile Met Tyr Asn Ile Leu Gly 370 375 380 Glu Asp Glu Gly Glu Ala Gly Phe Thr Gln Ala His Gln Leu Ile Glu 385 390 395 400 Arg Ala Leu Asp Ile Ser Gly Ala Ser Val His Trp Tyr Ala Lys Pro 405 410 415 Glu Ile Arg Lys Gln Arg Lys Met Gly His Ile Thr Ile Val Gly Pro 420 425 430 Ser Lys Tyr Ser Val Lys Ala Arg Leu Asp Lys Leu Leu Gln Arg Asp 435 440 445 Ala Tyr Asp Pro Lys Lys Val Ala Val Lys Pro Arg Ala Ala Ile Ile 450 455 460 Met Gly Ser Asp Ser Asp Leu Pro Val Met Lys Asp Ala Ala Val Val 465 470 475 480 Leu Lys Lys Phe Asn Ile Pro Phe Glu Leu Thr Ile Val Ser Ala His 485 490 495 Arg Thr Pro Glu Arg Met Tyr His Tyr Ala Leu Ser Thr Lys Glu Arg 500 505 510 Gly Leu Glu Val Ile Ile Ala Gly Ala Gly Gly Ala Ala His Leu Pro 515 520 525 Gly Met Val Ala Ser Leu Thr Ser Val Pro Val Ile Gly Val Pro Ile 530 535 540 Met Thr Ser Ser Leu His Gly Thr Asp Ser Leu Leu Ser Ile Val Gln 545 550 555 560 Met Pro Lys Gly Ile Pro Val Ala Thr Val Ala Ile Gly Asn Ala Glu 565 570 575 Asn Ala Gly Leu Leu Ala Val Arg Met Leu Ala Ser Arg Asp Pro Glu 580 585 590 Leu Gly Asp Lys Ala Thr Glu Tyr Gln His Asp Leu Arg Asp Met Val 595 600 605 Leu Glu Lys Ala Lys Arg Leu Glu Glu Leu Gly Trp Glu Glu Tyr Thr 610 615 620 Glu Leu Tyr Leu Lys Lys His 625 630 9 576 DNA Glycine max 9 gcacgagctg gtgctggtgg tgcagctcac ttgcctggta tggttgctgc ccttactccc 60 ttgcctgtta ttggcgttcc tgtgcgtgct tctaccttgg atgggattga ttcactcttg 120 tcaattgtcc agatgccgag aggtgtccct gttgccactg ttgcagttaa taatgcaact 180 aatgctggat tgctggcagt gaggatgttg ggtgttgcca atgataatct tctgtcaagg 240 atgagtcaat atcaagaggc ccaaaaggaa agcgtattgg gcaaaggaga taagttagaa 300 aaacatggct ggaaatccta cttaaacaat agttaattat ccccattaat ttggtgatta 360 ttttttaggc tcatttctca tttttggtca actacaaaat tttgatagaa aagatttcac 420 atggagggtg tatagaccca tggtatcaat aagtaggtta agaataattc gagctatatg 480 ttttgttacc taaatcaaca ggttttctat tttctattgc taacatatag caacagatga 540 aaattgtgta atctggcaaa aaaaaaaaaa aaaaaa 576 10 111 PRT Glycine max 10 Ala Arg Ala Gly Ala Gly Gly Ala Ala His Leu Pro Gly Met Val Ala 1 5 10 15 Ala Leu Thr Pro Leu Pro Val Ile Gly Val Pro Val Arg Ala Ser Thr 20 25 30 Leu Asp Gly Ile Asp Ser Leu Leu Ser Ile Val Gln Met Pro Arg Gly 35 40 45 Val Pro Val Ala Thr Val Ala Val Asn Asn Ala Thr Asn Ala Gly Leu 50 55 60 Leu Ala Val Arg Met Leu Gly Val Ala Asn Asp Asn Leu Leu Ser Arg 65 70 75 80 Met Ser Gln Tyr Gln Glu Ala Gln Lys Glu Ser Val Leu Gly Lys Gly 85 90 95 Asp Lys Leu Glu Lys His Gly Trp Lys Ser Tyr Leu Asn Asn Ser 100 105 110 11 348 DNA Triticum aestivum 11 ctttctgtaa ttgtggcaag gtgcagagat ggttgtacag tcggttatcc tgtcattgaa 60 accattcata aggataacat ctgtcatgtc gttgaagctc ctgctgagat acctgagaaa 120 atgaagaagt tagctaccaa tgtagctgaa aaaactatca aatcattaga aggtttatcg 180 aacgaagtag cccctagggc tcacattagt ggacaccata taatccagac atggcactct 240 tcacaatacg agcaacatct acgtgctatt cttggacttc ctgttggtaa ccctgtaatt 300 aaagcacccg caaccataat gaacaaaatc ctgggctatg atgaggtg 348 12 116 PRT Triticum aestivum 12 Leu Ser Val Ile Val Ala Arg Cys Arg Asp Gly Cys Thr Val Gly Tyr 1 5 10 15 Pro Val Ile Glu Thr Ile His Lys Asp Asn Ile Cys His Val Val Glu 20 25 30 Ala Pro Ala Glu Ile Pro Glu Lys Met Lys Lys Leu Ala Thr Asn Val 35 40 45 Ala Glu Lys Thr Ile Lys Ser Leu Glu Gly Leu Ser Asn Glu Val Ala 50 55 60 Pro Arg Ala His Ile Ser Gly His His Ile Ile Gln Thr Trp His Ser 65 70 75 80 Ser Gln Tyr Glu Gln His Leu Arg Ala Ile Leu Gly Leu Pro Val Gly 85 90 95 Asn Pro Val Ile Lys Ala Pro Ala Thr Ile Met Asn Lys Ile Leu Gly 100 105 110 Tyr Asp Glu Val 115 13 645 PRT Arabidopsis thaliana 13 Met Leu Leu Leu Lys Gln Ser Ser Ala Ala Val Leu Val Val Gly Asn 1 5 10 15 Thr Thr Pro Val Leu His Thr Ser Arg Ser Thr Tyr Arg Val Gly Pro 20 25 30 Phe Pro Val Thr Arg Thr Gln Ser Phe Gln Ser Leu Thr Met Ala Asn 35 40 45 Leu Gln Lys Leu Pro Thr Ser Ser Ser Gly Lys Leu Asn Thr Ala Ser 50 55 60 Ala Val Pro Cys Ser Ser His Asp Ala Ser Pro Ile Ser Glu Asn Arg 65 70 75 80 Glu Asn Lys His Val His Gly Val Ser Glu Lys Ile Val Gly Val Leu 85 90 95 Gly Gly Gly Gln Leu Gly Arg Met Leu Cys Gln Ala Ala Ser Gln Leu 100 105 110 Ala Ile Lys Val Met Ile Leu Asp Pro Ser Lys Asn Cys Ser Ala Ser 115 120 125 Ala Leu Ser Tyr Gly His Met Val Asp Ser Phe Asp Asp Ser Ala Thr 130 135 140 Val Glu Glu Phe Ala Lys Arg Cys Gly Val Leu Thr Val Glu Ile Glu 145 150 155 160 His Val Asp Val Asp Thr Leu Glu Lys Leu Glu Lys Gln Gly Val Asp 165 170 175 Cys Gln Pro Lys Ala Ser Thr Ile Arg Ile Ile Gln Asp Lys Tyr Met 180 185 190 Gln Lys Val His Phe Ser Gln His Gly Ile Pro Leu Pro Glu Phe Met 195 200 205 Glu Ile Ser Asp Ile Glu Gly Ala Arg Lys Ala Gly Glu Leu Phe Gly 210 215 220 Tyr Pro Leu Met Ile Lys Ser Lys Arg Leu Ala Tyr Asp Gly Arg Gly 225 230 235 240 Asn Ala Val Ala Asn Asn Gln Asp Glu Leu Ser Ser Ala Val Thr Ala 245 250 255 Leu Gly Gly Phe Ser Arg Gly Leu Tyr Ile Glu Lys Trp Ala Pro Phe 260 265 270 Val Lys Glu Leu Ala Val Ile Val Ala Arg Gly Arg Asp Gly Ser Met 275 280 285 Val Cys Tyr Pro Val Val Glu Thr Ile His Arg Asp Asn Ile Cys His 290 295 300 Ile Val Lys Ala Pro Ala Asp Val Pro Trp Lys Ile Asn Lys Leu Ala 305 310 315 320 Thr Asp Val Ala Gln Lys Ala Val Gly Ser Leu Glu Gly Ala Gly Val 325 330 335 Phe Ala Val Glu Leu Phe Leu Thr Glu Asp Ser Gln Ile Leu Leu Asn 340 345 350 Glu Val Ala Pro Arg Pro His Asn Ser Gly His Gln Thr Ile Glu Cys 355 360 365 Cys Tyr Thr Ser Gln Phe Glu Gln His Leu Arg Ala Val Val Gly Leu 370 375 380 Pro Leu Gly Asp Pro Ser Met Arg Thr Pro Ala Ser Ile Met Tyr Asn 385 390 395 400 Ile Leu Gly Glu Asp Asp Val Ile Asp Gly Glu Ala Gly Phe Lys Leu 405 410 415 Ala His Arg Leu Ile Ala Arg Ala Leu Cys Ile Pro Gly Ala Ser Val 420 425 430 His Trp Tyr Asp Lys Pro Glu Met Arg Lys Gln Arg Lys Met Gly His 435 440 445 Ile Thr Leu Val Gly Gln Ser Met Gly Ile Leu Glu Gln Arg Leu Gln 450 455 460 Cys Ile Leu Ser Glu Gln Ser His Gln Val His Glu Thr Pro Arg Val 465 470 475 480 Ala Ile Ile Met Gly Ser Asp Thr Asp Leu Pro Val Met Lys Asp Ala 485 490 495 Ala Lys Ile Leu Asp Leu Phe Gly Val Thr His Glu Val Lys Ile Val 500 505 510 Ser Ala His Arg Thr Pro Glu Met Met Tyr Thr Tyr Ala Thr Ser Ala 515 520 525 His Ser Arg Gly Val Gln Val Ile Ile Ala Gly Ala Gly Gly Ala Ala 530 535 540 His Leu Pro Gly Met Val Ala Ser Leu Thr Pro Leu Pro Val Ile Gly 545 550 555 560 Val Pro Val Arg Ala Thr Arg Leu Asp Gly Val Asp Ser Leu Leu Ser 565 570 575 Ile Val Gln Met Pro Arg Gly Val Pro Val Ala Thr Val Ala Ile Asn 580 585 590 Asn Ala Thr Asn Ala Ala Leu Leu Ala Val Arg Met Leu Gly Ile Ser 595 600 605 Asp Thr Asp Leu Val Ser Arg Met Arg Gln Tyr Gln Glu Asp Met Arg 610 615 620 Asp Glu Asn Leu Asn Lys Gly Glu Lys Leu Glu Thr Glu Gly Trp Glu 625 630 635 640 Ser Tyr Leu Asn Gln 645 14 623 PRT Nicotiana tabacum 14 Gly Pro Gln Arg Ser Ser Phe Ala Ser Pro Ile Leu Ala Val Asn Pro 1 5 10 15 Gln Lys Ser Ile Ser Phe Leu Lys Asn His Ser Phe Val Phe Ser Ser 20 25 30 Ser Leu Met Arg Gln Gln Ser Glu His Thr Pro Thr Met Leu Ser Cys 35 40 45 Lys Ala Ser Leu Glu Val Val Thr Asp Ser Pro Gly Gly Leu Glu Val 50 55 60 His Gly Ile Ser Glu Met Val Val Gly Val Leu Gly Gly Gly Gln Leu 65 70 75 80 Gly Arg Met Leu Cys Glu Ala Ala Ser Gln Met Ala Ile Lys Val Ile 85 90 95 Val Leu Asp Pro Met Asn Asn Cys Pro Ala Ser Ala Leu Ala His Gln 100 105 110 His Val Val Gly Ser Tyr Asp Asp Ser Ala Thr Val Glu Glu Phe Gly 115 120 125 Lys Arg Cys Gly Val Leu Thr Val Glu Ile Glu His Val Asp Val Val 130 135 140 Thr Leu Glu Lys Leu Glu Gln Gln Gly Val Asp Cys Gln Pro Lys Ala 145 150 155 160 Ser Thr Ile Arg Ile Ile Gln Asp Lys Tyr Leu Gln Lys Val His Phe 165 170 175 Ser Arg His Ala Ile Pro Leu Pro Lys Phe Met Gln Ile Asp Asp Leu 180 185 190 Glu Ser Ala Arg Arg Ala Gly Asp Leu Phe Gly Tyr Pro Leu Met Ile 195 200 205 Lys Ser Arg Arg Leu Ala Tyr Asp Gly Arg Gly Asn Ala Val Ala Lys 210 215 220 Ser Glu Glu Glu Leu Ser Ser Ala Val Asn Ala Leu Gly Gly Tyr Gly 225 230 235 240 Arg Gly Leu Tyr Val Glu Lys Trp Ala Pro Phe Val Lys Glu Leu Ser 245 250 255 Val Ile Val Pro Arg Gly Arg Asp Gly Ser Ile Ala Cys Tyr Pro Ala 260 265 270 Val Glu Thr Ile His Arg Asp Asn Ile Cys His Ile Val Lys Ser Pro 275 280 285 Ala Asn Val Ser Trp Lys Ser Met Lys Leu Ala Thr Asp Val Ala His 290 295 300 Arg Ala Val Ser Ser Leu Glu Gly Ala Gly Val Phe Ala Val Glu Leu 305 310 315 320 Phe Leu Thr Glu Asp Gly Gln Ile Leu Leu Asn Glu Val Ala Pro Arg 325 330 335 Pro His Asn Ser Gly His His Thr Ile Glu Ala Cys Phe Thr Ser Gln 340 345 350 Phe Glu Gln His Leu Arg Ala Val Val Gly Leu Pro Leu Gly Asp Pro 355 360 365 Ser Met Lys Thr Pro Ala Ala Val Met Tyr Asn Ile Leu Gly Glu Asp 370 375 380 Asp Gly Glu Pro Gly Phe Leu Leu Ala Asn Gln Leu Ile Glu Lys Ala 385 390 395 400 Leu Gly Ile Pro Gly Val Ser Val His Trp Tyr Asp Lys Pro Glu Met 405 410 415 Arg Arg Gln Arg Lys Met Gly His Ile Thr Ile Val Gly Pro Ser Met 420 425 430 Gly Ile Val Glu Ala Gln Leu Arg Val Ile Leu Asn Glu Glu Ser Val 435 440 445 Asn Gly His Pro Ala Val Ala Pro Arg Val Gly Ile Ile Met Gly Ser 450 455 460 Asp Ser Asp Leu Pro Val Met Lys Asp Ala Ala Lys Ile Leu Asn Glu 465 470 475 480 Phe Asp Val Pro Ala Glu Val Lys Ile Val Ser Ala His Arg Thr Pro 485 490 495 Glu Met Met Phe Ser Tyr Ala Leu Ser Ala Arg Glu Arg Gly Ile Gln 500 505 510 Val Ile Ile Ala Gly Ala Gly Gly Ala Ala His Leu Pro Gly Met Val 515 520 525 Ala Ala Phe Thr Pro Leu Pro Val Ile Gly Val Pro Val Arg Ala Ser 530 535 540 Thr Leu Asp Gly Leu Asp Ser Leu Leu Ser Ile Val Gln Met Pro Arg 545 550 555 560 Gly Val Pro Val Ala Thr Val Ala Ile Asn Asn Ala Thr Asn Ala Gly 565 570 575 Leu Leu Ala Val Arg Leu Leu Gly Ile Ser Asp Ile Lys Leu Gln Ala 580 585 590 Arg Met Ala Gln Tyr Gln Glu Asp Arg Arg Asp Glu Val Leu Val Lys 595 600 605 Gly Glu Arg Leu Glu Lys Ile Gly Phe Glu Glu Tyr Leu Asn Ser 610 615 620 15 557 PRT Vigna aconitifolia 15 Gly Leu Tyr Glu Val Val Val Gly Val Leu Gly Gly Gly Gln Leu Gly 1 5 10 15 Arg Met Met Cys Gln Ala Ala Ser Gln Met Ala Ile Lys Val Met Val 20 25 30 Leu Asp Pro Gln Glu Asn Cys Pro Ala Ser Ser Leu Ser Tyr His His 35 40 45 Met Val Gly Ser Phe Asp Glu Ser Thr Lys Val Glu Glu Phe Ala Lys 50 55 60 Arg Cys Gly Val Leu Thr Val Glu Ile Glu His Val Asp Val Asp Thr 65 70 75 80 Leu Glu Lys Leu Glu Lys Gln Gly Val Asp Cys Gln Pro Lys Ala Ser 85 90 95 Thr Val Arg Ile Ile Gln Asp Lys Tyr Gln Gln Lys Val Ala Leu Leu 100 105 110 Pro Ala Trp Ile Pro Leu Pro Glu Phe Met Lys Ile Asp Asp Leu Lys 115 120 125 Ala Lys Lys Trp Asp Ser Leu Asp Ile His Phe Met Ile Lys Ser Arg 130 135 140 Arg Leu Ala Tyr Asp Gly Arg Gly Asn Phe Val Ala Lys Ser Glu Glu 145 150 155 160 Glu Leu Ser Ser Ala Val Asp Ala Leu Gly Gly Phe Asp Arg Gly Leu 165 170 175 Tyr Ala Glu Lys Trp Ala Pro Phe Val Lys Glu Leu Ala Val Ile Val 180 185 190 Ala Arg Gly Arg Asp Asn Ser Ile Ser Cys Tyr Pro Val Val Glu Leu 195 200 205 Phe Thr Gly His Ile Cys His Ile Val Lys Ser Pro Ala Asn Val Asn 210 215 220 Trp Lys Thr Arg Glu Leu Ala Ile Glu Val Ala Phe Asn Ala Val Lys 225 230 235 240 Ser Leu Glu Val Pro Gly Val Phe Ala Val Glu Leu Phe Leu Thr Lys 245 250 255 Glu Gly Glu Ile Leu Leu Asn Glu Val Ala Pro Arg Pro His Asn Ser 260 265 270 Gly His His Thr Ile Glu Ser Cys His Thr Ser Gln Phe Glu Gln His 275 280 285 Leu Pro Ala Val Val Gly Leu Pro Leu Gly Asp Pro Ser Met Lys Thr 290 295 300 Pro Ala Ala Ile Met Tyr Asn Ile Leu Gly Glu Glu Glu Gly Glu His 305 310 315 320 Gly Phe Gln Leu Ala His Gln Leu Met Lys Arg Ala Met Thr Ile Pro 325 330 335 Gly Ala Ser Val His Trp Tyr Asp Lys Pro Glu Met Arg Lys Gln Arg 340 345 350 Lys Met Cys His Ile Thr Ile Val Gly Ser Ser Leu Ser Ser Ile Glu 355 360 365 Ser Asn Leu Ala Ile Leu Leu Glu Gly Lys Gly Leu His Asp Lys Thr 370 375 380 Ala Val Cys Ser Thr Leu Leu Gly Phe Ile Met Gly Ser Asp Ser Asp 385 390 395 400 Leu Pro Val Met Lys Ser Ala Ala Glu Met Met Glu Met Phe Gly Val 405 410 415 Pro His Glu Val Arg Ile Val Ser Ala His Arg Thr Pro Glu Leu Met 420 425 430 Phe Cys Tyr Ala Ser Ser Ala His Glu Arg Gly Tyr Gln Val Ile Ile 435 440 445 Ala Gly Ala Gly Gly Ala Ala His Leu Pro Gly Met Val Ala Ser Leu 450 455 460 Thr Pro Leu Pro Val Val Gly Val Pro Val Arg Ala Ser Thr Leu Asp 465 470 475 480 Gly Leu Asp Ser Leu Leu Ser Ile Val Gln Met Pro Arg Gly Val Pro 485 490 495 Val Ala Thr Val Ala Val Asn Asn Ala Thr Asn Ala Gly Leu Leu Ala 500 505 510 Val Arg Met Leu Gly Val Ala Asn Asp Asn Leu Leu Ser Arg Met Ser 515 520 525 Gln Tyr Gln Glu Asp Gln Lys Glu Ala Val Leu Arg Glu Gly Asp Lys 530 535 540 Leu Glu Lys His Gly Trp Glu Ser Tyr Leu Lys Asn Ser 545 550 555 16 2329 DNA Brassica 16 gtgagttaat gggttttgtt tccctcaaat aaaaatcgaa ccttcgaagc ttaagttctc 60 gcatcctctt tctccttgtc aagccacagc tcgaaaccag ctatgttcta gatgttgctt 120 ctgaaacaga gctcagctgc tgttcttgtc tctgggaatc caagtcctgt cctttacact 180 cctcgcttca cttccagagt tggatctctt ccagttagca aaacgaactc cttcaccatg 240 gcgaatcttc agaagggtct tacttattct tcttctgaga aattcaaccc ggtgttagcg 300 tgtagctctc acgaggcttc tcctatcagc gaggatacac atatcaaggg agtctctgag 360 atcattgtgg gagtgttggg aggtggacag ttaggtcgca tgctttgcca agctgcttct 420 caaatggcca tcaaggttat gattctagat ccttcaaaga actgttcagc aagctcatta 480 gcttatggcc acatggttga tagctttgac gacagtgcta cagttgaaga gtttgcaaaa 540 agatgtggag tcttgacagt agaaattgaa catgttgacg ttgaaacact agagaagctt 600 gagaaacaag gagtagatgt ccaaccaaaa gcctctacta tcaggataat acaggataaa 660 tacatacaaa aagttcattt ctctcggcat ggcatcccac ttccagagtt tatggagata 720 agcgatattg aaggagctga aagagcaggt gaactttttg gttaccctct tatgatcaag 780 agcaagagat tagcttatga tggacgagga aatgcagttg ctaatagcca agacgcgctt 840 acttctgctg taactgctct tggaggtttc agtcgtggtt tgtacgttga gaaatgggca 900 ccctttgtaa aggagttggc tgttattgtg gctaggggaa gagatggttc catggtttgt 960 tatccagttg ttgaaactgt tcacagggat aacatatgcc atatagttaa agcaccagca 1020 gatgtgcctt ggaagattaa caaacttgcc actgatgttg ctcaaaaggc tgttggttct 1080 ttagaaggcg ctggtgtttt cgctgttgag ctgttcttga cagaggatgg tcagatcctg 1140 ctgaatgaag ttgcacctag accacacaac agtggacatc agacgatcga gtcatgttac 1200 acttcacagt ttgagcaaca cttgcgagct gtggttggtc ttccactcgg tgatccgtct 1260 atgagaactc ctgcctccat tatgtacaat attctgggcg aagatgatgg agaagctggt 1320 ttcagattgg cacatcggct cattgcaaga gctctgagtg ttccaggtgc atctgtgcat 1380 tggtatgaca agccagaaat gagaaagcag cggaagatgg ggcacatcac tcttgttgga 1440 aagtctattg gtgttttgga acaaaggttg cattgtatat taagtgaaca aagccatcaa 1500 ctacatgaca ttgcagagat acctcgtgtt ggtatcatca tgggttcaga ctctgatctt 1560 cctgttatga aagatgctgc aaaaattctt gacacgttta atgtaacata tgaggtgaag 1620 atagtttcag cacatcggac accagagatg atgttttctt atgcaacatc agctcatagt 1680 agaggaatcc aagtgataat tgcaggtgct ggtggtgctg ctcacttacc aggtatggtt 1740 gcttcactca ctcccttacc tgtgattggt gtccctgtac gtgctacccg tttggatgga 1800 gttgattcac ttctctccat tgttcagatg cctagaggtg ttcctgtagc cacagttgct 1860 ataaacaact ccaccaacgc agccttgctt gctatcagga tgctggggat ctctgatact 1920 gatctcgtct caaggataag tcagtaccag gaagacatga gagaagagaa catggttaaa 1980 ggtgagaaac ttgagcgtca aggttgggaa tcatacttga accagtgaat attacttgtc 2040 agaactcctc attggttacc gggaactcca tagtgatcga ttttgctgcc agtaagatct 2100 tgttgagagt tagtttcgga tcttgagttg ttgtcacggc taattccggt ttagctttgt 2160 caagttaagc cacaattgtt tgagactcaa gctaagtcct ggtgagggat ctgatagtta 2220 gcttcagttc ttgagaaact attggatgag ttatccacac gatgttttat tttataatat 2280 tatgtaactc ccatagagac atgggatata acatgttctt acaaaaaaa 2329 17 638 PRT Brassica 17 Met Leu Leu Leu Lys Gln Ser Ser Ala Ala Val Leu Val Ser Gly Asn 1 5 10 15 Pro Ser Pro Val Leu Tyr Thr Pro Arg Phe Thr Ser Arg Val Gly Ser 20 25 30 Leu Pro Val Ser Lys Thr Asn Ser Phe Thr Met Ala Asn Leu Gln Lys 35 40 45 Gly Leu Thr Tyr Ser Ser Ser Glu Lys Phe Asn Pro Val Leu Ala Cys 50 55 60 Ser Ser His Glu Ala Ser Pro Ile Ser Glu Asp Thr His Ile Lys Gly 65 70 75 80 Val Ser Glu Ile Ile Val Gly Val Leu Gly Gly Gly Gln Leu Gly Arg 85 90 95 Met Leu Cys Gln Ala Ala Ser Gln Met Ala Ile Lys Val Met Ile Leu 100 105 110 Asp Pro Ser Lys Asn Cys Ser Ala Ser Ser Leu Ala Tyr Gly His Met 115 120 125 Val Asp Ser Phe Asp Asp Ser Ala Thr Val Glu Glu Phe Ala Lys Arg 130 135 140 Cys Gly Val Leu Thr Val Glu Ile Glu His Val Asp Val Glu Thr Leu 145 150 155 160 Glu Lys Leu Glu Lys Gln Gly Val Asp Val Gln Pro Lys Ala Ser Thr 165 170 175 Ile Arg Ile Ile Gln Asp Lys Tyr Ile Gln Lys Val His Phe Ser Arg 180 185 190 His Gly Ile Pro Leu Pro Glu Phe Met Glu Ile Ser Asp Ile Glu Gly 195 200 205 Ala Glu Arg Ala Gly Glu Leu Phe Gly Tyr Pro Leu Met Ile Lys Ser 210 215 220 Lys Arg Leu Ala Tyr Asp Gly Arg Gly Asn Ala Val Ala Asn Ser Gln 225 230 235 240 Asp Ala Leu Thr Ser Ala Val Thr Ala Leu Gly Gly Phe Ser Arg Gly 245 250 255 Leu Tyr Val Glu Lys Trp Ala Pro Phe Val Lys Glu Leu Ala Val Ile 260 265 270 Val Ala Arg Gly Arg Asp Gly Ser Met Val Cys Tyr Pro Val Val Glu 275 280 285 Thr Val His Arg Asp Asn Ile Cys His Ile Val Lys Ala Pro Ala Asp 290 295 300 Val Pro Trp Lys Ile Asn Lys Leu Ala Thr Asp Val Ala Gln Lys Ala 305 310 315 320 Val Gly Ser Leu Glu Gly Ala Gly Val Phe Ala Val Glu Leu Phe Leu 325 330 335 Thr Glu Asp Gly Gln Ile Leu Leu Asn Glu Val Ala Pro Arg Pro His 340 345 350 Asn Ser Gly His Gln Thr Ile Glu Ser Cys Tyr Thr Ser Gln Phe Glu 355 360 365 Gln His Leu Arg Ala Val Val Gly Leu Pro Leu Gly Asp Pro Ser Met 370 375 380 Arg Thr Pro Ala Ser Ile Met Tyr Asn Ile Leu Gly Glu Asp Asp Gly 385 390 395 400 Glu Ala Gly Phe Arg Leu Ala His Arg Leu Ile Ala Arg Ala Leu Ser 405 410 415 Val Pro Gly Ala Ser Val His Trp Tyr Asp Lys Pro Glu Met Arg Lys 420 425 430 Gln Arg Lys Met Gly His Ile Thr Leu Val Gly Lys Ser Ile Gly Val 435 440 445 Leu Glu Gln Arg Leu His Cys Ile Leu Ser Glu Gln Ser His Gln Leu 450 455 460 His Asp Ile Ala Glu Ile Pro Arg Val Gly Ile Ile Met Gly Ser Asp 465 470 475 480 Ser Asp Leu Pro Val Met Lys Asp Ala Ala Lys Ile Leu Asp Thr Phe 485 490 495 Asn Val Thr Tyr Glu Val Lys Ile Val Ser Ala His Arg Thr Pro Glu 500 505 510 Met Met Phe Ser Tyr Ala Thr Ser Ala His Ser Arg Gly Ile Gln Val 515 520 525 Ile Ile Ala Gly Ala Gly Gly Ala Ala His Leu Pro Gly Met Val Ala 530 535 540 Ser Leu Thr Pro Leu Pro Val Ile Gly Val Pro Val Arg Ala Thr Arg 545 550 555 560 Leu Asp Gly Val Asp Ser Leu Leu Ser Ile Val Gln Met Pro Arg Gly 565 570 575 Val Pro Val Ala Thr Val Ala Ile Asn Asn Ser Thr Asn Ala Ala Leu 580 585 590 Leu Ala Ile Arg Met Leu Gly Ile Ser Asp Thr Asp Leu Val Ser Arg 595 600 605 Ile Ser Gln Tyr Gln Glu Asp Met Arg Glu Glu Asn Met Val Lys Gly 610 615 620 Glu Lys Leu Glu Arg Gln Gly Trp Glu Ser Tyr Leu Asn Gln 625 630 635 

What is claimed is:
 1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having phosphoribosylaminoimidazole carboxylase activity, wherein the polypeptide has an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or 17, or (b) a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
 2. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide has at least 85% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 3. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide has at least 90% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 4. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide has at least 95% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 5. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide comprises one of SEQ ID NO:2, 4, 6, 8 or
 17. 6. The polynucleotide of claim 1 wherein the nucleotide sequence comprises one of SEQ ID NO:1, 3, 5, 7 or
 16. 7. A vector comprising the polynucleotide of claim
 1. 8. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
 9. A method for transforming a cell, comprising transforming a cell with the polynucleotide of claim
 1. 10. A cell comprising the recombinant DNA construct of claim
 8. 11. A method for producing a plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.
 12. A plant comprising the recombinant DNA construct of claim
 8. 13. A seed comprising the recombinant DNA construct of claim
 8. 14. An isolated polypeptide having phosphoribosylaminoimidazole carboxylase activity, wherein the polypeptide has an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 15. The polypeptide of claim 14, wherein the amino acid sequence of the polypeptide has at least 85% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 16. The polypeptide of claim 14, wherein the amino acid sequence of the polypeptide has at least 90% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 17. The polypeptide of claim 14, wherein the amino acid sequence of the polypeptide has at least 95% sequence identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO:2, 4, 6, 8 or
 17. 18. The polypeptide of claim 14, wherein the amino acid sequence of the polypeptide comprises one of SEQ ID NO:2, 4, 6, 8 or
 17. 19. A method for isolating a polypeptide having phosphoribosylaminoimidazole carboxylase activity comprising isolating the polypeptide from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
 20. A method of altering the level of expression of a phosphoribosylaminoimidazole carboxylase in a host cell comprising: (a) transforming a host cell with the recombinant DNA construct of claim 8; and (b) growing the transformed host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of the phosphoribosylaminoimidazole carboxylase in the transformed host cell.
 21. A method for evaluating at least one compound for its ability to inhibit phosphoribosylaminoimidazole carboxylase activity, comprising the steps of: (a) introducing into a host cell the recombinant DNA construct of claim 8; (b) growing the host cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of a phosphoribosylaminoimidazole carboxylase; (c) optionally purifying the phosphoribosylaminoimidazole carboxylase expressed recombinant DNA construct in the host cell; (d) treating the phosphoribosylaminoimidazole carboxylase with a compound to be tested; (e) comparing the activity of the phosphoribosylaminoimidazole carboxylase that has been treated with a test compound to the activity of an untreated phosphoribosylaminoimidazole carboxylase, and (f) selecting compounds with potential for inhibitory activity. 